Following the widespread use of antibiotics and immunosuppressive therapy over the past 30 years, there has been a dramatic rise in fungal disease. There is an urgent need to identify molecules which are unique to fungi and may be utilized as drug targets. Lysine is the only amino acid which is synthesized by the alpha- aminoadipate pathway, unique to fungi and not present in bacteria, plants, animals, or humans. The pathway consists of eight enzyme steps and more than ten unlinked genes in Saccharomyces cerevisiae. This pathway is also present in pathogenic fungi, including the most important opportunistic yeast pathogen Candida albicans. One of the central enzymes of this pathway, alpha-aminoadipate reductase (AAR), catalyzes the conversion of alpha-aminoadipate to alpha-aminoadipate- semialdehyde in the presence of ATP, MgCl2, and NADPH, through a complex adenylation of the substrate alpha-aminoadipate by ATP and subsequent reduction by NADPH. This 180 kDa heterodimeric enzyme is encoded by two distinct genes, LYS2 and LYS5 of S. cerevisiae in which LYS2 encodes the large approximately 150 kDa subunit and LYS5 encodes the small approximately 30 kDa subunit. The 4.2 kb C. albicans LYS2 open reading frame also encodes a approximately 150 kDa polypeptide consisting of 1391 amino acid residues. Sequence comparison of the encoded Lys2p revealed a strong conservation of functional domains unique to a super family of proteins that activates amino acids to acyl-AMP derivatives (such as the nonribosomal peptide antibiotic ACV synthetase which also uses alpha-aminoadipate as a substrate). The objective of this prooject is to determine the molecular mechanism of adenylation of alpha-aminoadipate and the structure function relationship of the LYS2 gene and Lys2p of C. albicans. A specifically designed genomic disrupted lys2 mutant, purified recombinant Lys2p, and site-directed mutations of the LYS2 gene will be produced and analyzed to address the fundamental questions of physical properties and adenylation activity of Lys2p. Recombinant Lys2p will be expressed and purified using an Escherichia coli expression system and investigated for molecular and kinetic properties as well as the AAR, adenylation and NADPH oxidation activities. All isogenic knockout lys2 mutant will be produced through sequential disruption of both alleles of the LYS2, and site-directed mutations at conserved amino acid residues will be obtained by primer mutagenesis of the LYS2 gene. These mutants will be employed in in vivo transformation experiments and in vitro enzyme assays to determine the specific physiological roles of the mutated amino acid residues in the complex AAR reaction.